Introduction

What is Biotechnology? - Purposeful design and modification/assembly of bio-oriented materials (e.g.,

proteins/enzymes, microorganisms, plant/animal cells, tissues, stem cells etc..)

and unit processes to benefit humans or make a profit.

- Use and applications of biological system (cells, tissues etc..) or biomolecules (enzymes/proteins, antibodies, DNA/RNA) and key technologies to produce

valuable products at commercial scale and to treat diseases:

Cost-effectiveness economically feasible

Basic Biology / Medical sciences - To discover and understand the underlying mechanisms of behaviors

and disorders in living organisms

Traditional Biotechnology (Before 1970) - Broad definition of Biotech : Using a biological system to make products

- Food processing : Fermented foods, Brewery, Dairy products, etc.

Biological process of brewing beer : conversion of starch to sugar followed

by addition of specific yeast

- Agriculture : Modifications of living plants for improved yield of food crops via

artificial selection and hybridization: Breeding

ex) Crops with reduced vulnerability to frost, draught, and the cold

Simple process

- Direct use of or isolation from original biological sources

- Fermentation: production of acetone using Clostridium acetobutylicum

Definition of Biotechnology based on the use of techniques/methods

• Use of recombinant DNA technology since 1973

- Cohen and Boyer : gene manipulation techniques to cut and paste DNA

(using restriction enzymes and ligases) and transfer the new DNA into bacteria.

Revolutionize traditional biotechnology

• Combined use of different disciplines:

- Biology-based knowledge : Cell biology, genetics, molecular biology, etc

- Knowledge linked with practical applications :Biochemical Eng, Bioinformatics,

computational design, Organic chemistry etc.

• Use of genetically engineered microorganisms

- Enabling the production of existing medicines or products easily and cheaply

(ex: Insulin (51 amino acids) : discovered by Banting and Macleod from Univ. of Toronto, awarded Nobel Prize in 1923. Assistants : Charles Best (not awarded)

- First genetically engineered synthetic insulin (Humulin) by E. coli in 1982)

• Traditional Biotechnology industries : adopts new approaches and modern techniques

to improve the quality and productivity of high value-added products

Modern Biotechnology (After 1970s)

Impact of recombinant DNA technology on the production of proteins

• Overcomes the problem of source availability : allows the manufacture of any protein in whatever quantity it is required

• Overcomes the problem of product safety:

Transmission of blood-born pathogens such as hepatitis B, C, and HIV

via infected blood products

• Provides an alternative to direct extraction from inappropriate or dangerous source materials : The fertility hormones( FSH and hCG) from the urine of pregnant women; Urokinase from urines

• Facilitates the generation of newly designed proteins:

Therapeutic proteins or enzymes with desired property

• Development of therapeutics based on underlying mechanisms of diseases

- Development of new methods to cure diseases : Gene and cell (stem cells) therapies, therapeutic proteins

• Production of valuable products at commercial scale

Organic acids, Antibiotics, Amino acids, Proteins(enzymes), Biofuels, Vitamins,

Hormones, Alcohols, Fermented foods, Fine chemicals, etc..

• Development of tools and methodology Expression systems, Gene synthesis/Sequencing, Genome editing, Diagnosis,

Delivery, Purification process, Formulation, Bioassays

Major focus of Biotechnology

Integration of biological sciences with Engineering principles

cost-effectiveness

Required disciplines

- Biology

- Physical, organic chemistry / Pharmacology, Electronics

- Biochemical engineering : Extension of chemical engineering principles to biological system Mass/Heat/Energy transfer, - Thermodynamics Bioreaction engineering, plant design, process control / optimization, and separations

Basic Biology

Biotechnology Bio-industry - Pharmaceutical - Biotech. company Engineering principles

Biotechnology is a multi-disciplinary field

Recombinant E. coli

G protein-coupled receptor(GPCR)

DNA microarray

Gene therapy using adenovirus Nanobiotechnology

• Health care / Diagnostics : - Development of therapeutics: efficacy, toxicity - Diagnosis : early detection and prevention of diseases

• Agriculture : Crop production with high yield and quality • Bio-based process: Pollution, CO2 emission, global warming • Alternative energy (Bio-energy) : - Depletion of fossil fuels - Use of renewable sources :Corn, sugar cane, cellulose - Cost (?)

Major application areas

• Protein engineering : Design of proteins/enzymes based on structural and mechanistic knowledge, molecular evolution, computational design

• Metabolic pathway engineering: Design of more efficient metabolic pathways:

high yield of target product, low by-product

• Computational modeling and optimization: Systems Biology, Genome- and

proteom-wide analyses

• Nano-biotechnology : Integration of nanotechnology

- Use of NPs for diagnosis, drug delivery, and imaging

- Nano-medicine

Key fields

• Cell culture engineering : Cultivation of microorganisms and mammalian cells

- Hybridoma technology : A technology of forming hybrid cell lines (called hybridoma) by fusing a specific antibody-producing B cell with a myeloma

(B cell cancer) cell that is selected for its ability to grow in culture media.

• Tissue engineering : Use of a combination of cells, engineering and materials/ methods, and suitable biochemical and physio-chemical factors to repair or replace portions of or whole tissues (i.e., bone, cartilage, blood vessels, bladder, skin, muscle etc,--> artificial organs )

• Synthetic biology : Designing and constructing biological devices and bio

logical systems for useful purposes.

- Creation of new bio-systems (Cells and biomolecules): Systematic, hierarchical

design of artificial, bio-inspired system using robust, standardized and well-

characterized building blocks or parts

• Separation technology : Recovery and purification of a target product

Branches of Biotechnology

• Blue biotechnology : Marine and aquatic applications of biotechnology

• Green biotechnology : Agricultural applications

Plant biotechnology

• Red biotechnology : Medical applications

Nanomedicine, Regenerative medicine

• White biotechnology : Industrial applications

- Production of bio-chemicals using bioprocess

New paradigms in Biotechnology

• Massive and high-speed analysis system

- Genome and proteom-wide approach : Systemic approach

- Huge amounts of relevant knowledge,

• Genomics (Gene chips) : Sequences of more than few hundreds genomes

- 1 million genes / chip

- Gene (mRNA) expression profiling in high throughput way

- Single nucleotide polymorphism (SNP)

• Proteomics (2-D gel, LC/MS, protein microarray)

- Functional genomics

- Bio-molecular interactions (Interactoms)

• Development and commercialization of target products

- Bioinformatics

Genome- and proteom-wide analyses: Global analysis Integration of high-throughput analysis system

Bio-based economy: Impact on global economy

Shift from petroleum-based economy - Exhaustion and soaring price of petroleum (> $ 100 /gallon)

- Environmental issue

Global warming (greenhouse gas, CO2 , emission)

Pollution

• Development of renewable source-based Bioprocess

• Replacement of chemical processes with Bio-based ones

White Biotechnology

Company Products

BASF

Vitamin B-2 Methoxy isopropyl amine (chiral intermediate) Styrene oxide Amino acids

Eastman Chemical / Genencor Ascorbic acid

Degussa

Acrylamide Fatty acid – derived esters Polyglycerine ester Organo modified silicones and oleochemicals

Celanese / Diversa

Acetic acid Polyunsaturated fatty acids Non-digestible starch Polylactic acid (PLA)

Cargill Polylactic acid (PLA) (140,000 MT/yr)

DuPont / Genencor 1,3-Propanediol Terephthalic acid Adipic acid

Chevron / Maxygen Methanol

Typical examples of Bio-Products

Cell culture

Bioconversion

Feedstock Bioprocessing Product

GAS

LIQUID

SOLID

PRODUCT

LINES

Biocatalyst

Cells Bioreactor

Recovery

product

Feedstock Bioprocessing

Gas − Syn. Gas − CO2

− Organic vapor

Liquid − Organic − Sugar solution

Solid − Biomass − Consumer Waste

Bioconversion

by enzymes − Ambient to Extreme

Cell culture − Bacteria/yeast −Mammalian cells − Ambient to Extreme

Bioreactors − Continuous Systems − Membrane − Batch or Fed-batch

Media - Aqueous - Organic solvent

Separation /purification − In situ − Secondary

Pharmaceuticals Fine chemicals Specialty Chemicals Feedstock Bulk chemicals

General scheme for bioprocess

Products

Value chains from renewable sources

Alternative energy sources

Production of biofuels from renewable sources

• Increase in the yield and alcohol tolerance - Redesign of pathway for the ethanol production in yeast to use raw materials :

corn starch, cellulose, soybean, sugar cane

- Elucidation of enzyme mechanisms

- Redesign of pathway to increase the yield and to reduce by-products

- Design of critical enzymes in the pathway

• Process development : Fermentation process

• Separation and concentration

Role of Agricultural Biotech in the production of biofuels ? Adverse effects due to the production of biofuels from corn ?

Enzymes play a key role in Bio-based economy

Energy and Environmental issues - Depletion of fossil fuels - Limitation to CO2 emission (Kyoto protocol)

Renewable source-based economy

Bio-based process

Enzymes

Petrochemical-based economy

Chemical process

Use of enzymes for biofuel and biochemicals from renewable biomass such as starch and cellulose amylase, cellulase etc.

Biomolecular Eng. Lab.

Enzymes : Biocatalysts

- Cleaning (Detergents) - Textiles - Starch Processing - Leather - Baking - Pulp and Paper - Food and Specialties - Cosmetics

Most proficient catalysts with high specificity Competitive and cost-effective processes

Chiral drugs

Chiral intermediates

Semisynthetic antibiotics

Organic acids

Synthesis of specialty chemicals

Use for biosciences

DNA polymerase: Thermostability, fidelity

Restriction enzymes: Specificity

Alkaline phosphatase, Peroxidase

Use for daily life

Chemical company devoting to Biotechnology : BASF

Ecoflex®

Emphasis on Bio-products mainly using enzymes

Therapeutic proteins

Small molecule-based drugs : Efficacy, side effect, safety

Therapeutic proteins : High efficacy and safety, less toxicity

- Antibodies, proteins, enzymes, peptides etc.

ex) EPO, Interferon, Insulin, Avastin, Enbrel, Remicade, Herceptin,

EPO (Erythropoietin) : Stimulating the proliferation of red blood cells

Herceptin : Mab against EGFR2(Epidermal growth factor receptor 2) Avastin : Mab against VEGF (Vascular endothelial growth factor) Remicade: Mab against TNF-α (Tumor necrosis factor- α)

World market

- EPO alone : ~ $ 11 billion per year

- Remicade : ~ $ 9 billion per year

- $ 50 Billion (2007) $ 190 Billion (2015)

- Intensive investment in monoclonal antibodies: Biosimilar

Therapeutic proteins will form the back-born of future biotech market

Brand name Active ingredient

Type Class Treatment Company 2013 global sales (US$ billion)

Patent expiry EU/US

Humira adalimumab Antibody TNF inhibitor Arthritis Abbott/Eisai 10.7 Apr 2018/ Dec 2016

Remicade infliximab Antibody TNF inhibitor Arthritis Merck/Mitsubishi 8.9 Aug 2014/ Sep 2018

Rituxan rituximab Antibody Anti-CD20 Arthritis, NHL Roche/Biogen 8.6 Nov 2013/ Dec 2018

Enbrel etanercept Antibody TNF inhibitor Arthritis Amgen/Pfizer 8.3 Feb 2015/ Nov 2028

Lantus insulin glargine Protein Insulin receptor Diabetes Sanofi 7.8 2014/2014

Avastin bevacizumab Antibody Anti-angiogenesis Cancer Roche 7.0 Jan 2022/ Jul 2019

Herceptin trastuzumab Antibody Anti-HER2 Breast cancer Roche 6.8 Jul 2014/ Jun 2019

Neulasta pegfilgrastim Protein G-CSF Neutropenia Amgen 4.4 Aug 2017/ Oct 2015

Top 8 blockbuster biologicals (2013)

1 2

3 3

2

1 CDRS

FR

VL VH

Structural and functional features of antibodies

• Complicated process for selecting cell lines and

the production using mammalian cells very expensive

• Intellectual property barriers

• Tend to aggregate due to large size (~ 150 KDa)

• Difficult to penetrate inside the cells

• Limited binding affinity due to confined binding surface

Non-antibody scaffold to replace antibodies

High-level soluble expression in bacteria

High stability (thermodynamic, pH)

Easy design of binders with high affinity for a target

Low immunogenicity and cytotoxicity

Drawbacks of immunoglobulin antibodies

Therapeutics based on non-antibody scaffold

Development of new therapeutics with high efficacy and low side effects

from non-antibody protein scaffolds

Designer therapeutic proteins : Specificity and binding affinity

IP issue and cost-effectiveness

GlaxoSmithKline, Amgen Bristol-Myers-Squibb, Boehringer Ingelheim Eli Lilly, Roche, Avidia, Ammunex. Affibody, Ablynex,

Adnexus Therapeutics …….

Strategic alliance or merger between big pharma and biotech companies

New paradigm in therapeutic proteins

- Technology and idea - Financial investment

Non-antibody scaffolds

Human fibronectin Human lipocalin

Z domain of Staphylococcal protein A

Ankyrin Repebody

Disease Product name

Developer

Sales (US$Millions) Features 2004 2007

Gaucher’s

Ceredase® Genzyme 443 N/A Glucocerebrosidase (β-Glucosidase)

Purified from human placenta

Cerezyme® Genzyme 932

(2005) 1,048

Produced in CHO cells

3 Exoglycosidases process for Terminal Mannose

Fabry’s Fabrazyme® Genzyme 209 397 α-galactosidase

Mannose-6-phosphate for Glycotargeting Replagal TKT 57 168

MPS-1 Aldurazyme® Genzyme 12 204 α –L-iduronidase

Pompe Myozyme® Genzyme Approved

(2006) α-glucosidase

Therapeutic Enzymes : Enzyme replacement treatment

Treatment of Gaucher’s disease by Cerezyme costs up to $550,000 annually: Orphan drug and life-long treatment

Most of therapeutic enzymes : glycoproteins

Glucocerebrosidase (β-Glucosidase)

- Found by Phillipe Gaucher in 1882

- Biochemical basis for the disease in 1965 by Brady et al..

Glucosyl

CH2-CH-CH-CH=CH-(CH2)12-CH3

O=C-CH2-CH2-CH2-(CH2)n-CH3 N OH

Ceramide

OH-CH2-CH-CH-CH=CH-(CH2)12-CH3

O=C-CH2-CH2-CH2-(CH2)n-CH3 N OH

Glucose Ceramide

Gaucher’s Disease : Lysosomal Storage Disease

Autosomal recessive inheritance

- Caused by a recessive mutation in a gene located on chromosome 1, affecting both males and females

- Most common of the LSD

Glucocerebroside: Constituent of red and white blood cell membranes

Lysosomal storage diseases (LSDs): Lysosomal Enzymes

Lysosomes: Cellular organelles containing acid

hydrolase enzymes to break down waste materials and cellular debris

Cells’ garbage disposal system • Digestive organelle in the cell • Contains ~40 hydrolytic enzyme • Acidic pH (about pH4.8) within the

lysosome : the activity of lysosomal enzymes

(1) The ER and Golgi apparatus make a lysosome

(2) The lysosome fuses with a digestive vacuole

(3) Activated acid hydrolases digest the contents

(LSD) Lysosome

Nucleus

Mitochondria

Lysosome with substrate accumulation

(Normal cell) (LSD cell)

Normal cells Glucocerebrosides

Glucocerebrosides

Digestive vacuole

Gaucher cells

Digestive vacuole

Glucocerebrosidase

Incomplete digestion

Exocytosis

Residual vacuole

glucose ceramide +

Residual vacuole accumulated

No exocytosis

1/ 40,000~60,000 (Jew 1/~500) Swollen vacuoles Gaucher cells Accumulation in spleen, liver, kidney, brain Enlarged spleen and liver, liver malfunction, neurological complications etc..

Gaucher’s disease : Occurrence and symptoms

Distended abdomen

Diagnostics

• Diagnosis of disease as early as possible :

Best solution compared to treatments

• Prediction and treatment of diseases based on individual

genome sequence

- Personalized medicine

- Treatment with appropriate therapeutic agents

• Analysis / Detection of disease biomarkers:

- Invasive or non-invasive analysis

- Isolation and analysis of circulating tumor cells and DNAs

• Integration with nanotechnology : Sensitivity and accuracy

Regenerative medicine

Translational research for replacing, engineering or regenerating human cells, tissues or organs to restore or establish normal function

Genome editing • A type of gene engineering in which DNA is inserted, replaced, or removed from a

genome using artificially engineered nucleases, or "molecular scissors."

• Four families of engineered nucleases

- Meganucleases: Endodeoxyribonucleases with a large recognition site

(double-stranded DNA sequences of 12 to 40 base pairs)

- Zinc finger nucleases (ZFNs)

- Transcription Activator-Like Effector Nucleases (TALENs)

- CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas system

Biotechnology will have the greatest impact on humans

in the future in terms of health care, life-style, and economy.

- Therapeutic proteins

- Bio-based economy : High-value compounds by bioprocess

- Diagnostics

Modern Biotechnology constitutes a variety of diverse areas

and technologies, requiring interdisciplinary collaborations.

Perspectives